Not for Publication. 2 Evaluating an Experimental Dentifrice Containing. 3 Chloramine-T on Oral Health: A Preliminary Study

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1 1 Publication ORIGINAL ARTICLE 2 Evaluating an Experimental Dentifrice Containing 3 Chloramine-T on Oral Health: A Preliminary Study 4 Camila Tirapelli a /Flávio Landi b /José Paulo Ribas a /Heitor Panzeri a / 5 Elza Helena Guimarães Lara b 6 Purpose: The aim of the present study was to evaluate the clinical efficacy of an experimental dentifrice (CH) containing 7 an antimicrobial agent (1% Chloramine-T). 8 9 Materials and Methods: A clinical, fully randomised, double-blind comparative study was designed 30 selected 10 patients aged 15 to 50 years, with no periodontal disease, decay or other oral diseases, good general health and the 11 presence of dental plaque and sulcus gingival bleeding. Baseline Turesky modified plaque index (PI) and sulcus gingival 12 bleeding index (SBI) were scored all patients. Volunteers randomly received the experimental product (CH) or a 13 commercial-brand dentifrice containing triclosan (TR). Both dentifrices were provided in identical, number-labelled tubes, 14 and the subjects were instructed to use the supplied dentifrice only their usual oral hygiene, three times a day a 15 duration of 7 days. After 7-day use of dentifrices, the PI and SBI were assessed again. The data obtained were subjected 16 to the Kruskal Wallis test, followed Dunn s post hoc test Results: After 7-day use of dentifrices, the PI scores diminished significantly both evaluated dentifrices. The SBI 19 score values decreased significantly both experimental and commercial-brand dentifrices Conclusions: Both dentifrices reduced PI and SBI. By comparing the experimental and gold-standard dentifrice, it was 22 found that there was no statistical difference between the PI and SBI scores after their use, suggesting that they exerted 23 a similar effect on the oral health indexes. 24 Key words: clinical trial, dentifrices, development of mulations, oral health 25 Oral Health Prev Dent 2010; 8: xxx xxx. Submitted publication: ; accepted publication: D 28 espite improvements in oral health status all 29 over the world (Glass, 1982), dental caries and 30 periodontal disease are still considered the two most 31 prevalent oral diseases (Socransky and Haffajee, ). In dentistry, preventive oral care includes spe- 33 cific strategies to prevent oral diseases (primary pre- 34 vention) or to stop/treat disease progression in its a Department of Dental Materials and Prosthesis, School of Dentistry, University of São Paulo, Avenida do Café, s/n, Ribeirão Preto, São Paulo, Brazil. b HELP Laboratory of Research and Development, Avenida Meira Junior, 430. Ribeirão Preto, São Paulo, Brazil. Correspondence: Camila Tirapelli, Department of Dental Materials and Prosthesis, Faculty of Dentistry of Ribeirão Preto, University of São Paulo, Avenida do Café, s/n, , Ribeirão Preto, São Paulo, Brazil. Tel: Fax: catirapelli@p.usp.br initial stage (secondary prevention). As preventive dentistry is less invasive, traumatic and expensive and, in the long term, more effective than the interventional procedures, many investigators have directed their considerable efts towards developing tools and knowledge to improve the effectiveness and prevalence of these prevention measures. Preventive oral care associated with caries, gingivitis and periodontal disease includes the control of dental biofilm on the teeth and the adjacent gum area. The safest individual method to control dental biofilm is mechanical removal (Axelsson et al, 1991). In this context, it is important to remember the assertion of Costerton and Lashen (1984) that there is no effective chemical means removing dental biofilm. Mechanical control of dental biofilm is intrinsically related to toothbrushes and dentifrices (Forward, 1991); their ease of use and relatively low cost are Vol 8, No 4,

2 Tirapelli et al 1 the important characteristics their worldwide 2 acceptance as two efficient oral health tools (Pader, ). In Brazil, instance, the government 4 considers dentifrices as one of the items essential 5 family use, along with salt, oil, flour, soap and 6 others. 7 Today, with the development of technology and 8 the unabated search better health conditions, 9 dentifrices serve as a complementary product 10 the mechanical control of dental plaque; a great 11 number of innovative mulations have been pro- 12 posed in a preventive approach (Tirapelli et al, ). Furthermore, dentifrices have become vehi- 14 cles a wide variety of chemical antimicrobial 15 agents, such as triclosan (Triratana et al, 2002; 16 Davies et al, 2004); chlorhexidine (Olympio et al, ; Slot et al, 2007); medicinal plants (Panutti 18 et al, 2003; Singh et al, 2007; Alviano et al, ); Aloe vera (Oliveira et al, 2008); lotus leaf 20 (Li and Xu, 2007) and chlorine compounds (Panzeri 21 et al, 2008). The required characteristics a chem- 22 ical agent to effectively control dental biofilm were 23 postulated Loescher in 1976, which include sub- 24 stantivity, stability and safety (Gaffar et al, 1997). 25 The antimicrobial agent proposed in this clinical 26 study was Chloramine-T, which has already been 27 studied its use in mouthrinses (Pitten and 28 Kramer, 1999) and in a dentifrice total denture 29 cleaning (Panzeri et al, 2008). Chloramine-T has 30 the following characteristics: Effectiveness against microorganisms: Chlora- 34 mine-t is a salt named N-sodium-N-chloro-p-tolu- 35 enesulphonamide. As an N-chloro compound, it 36 contains active (electrophilic) chlorine com- 37 pounds (Gottardi, 1992), such as NaClO and 38 HClO (Hahn et al, 1994). These active chlorine 39 derivatives from Chloramine-T have been used 40 as disinfectants in algaecides, bactericides and 41 germicides parasite control and drinking 42 water disinfection. Its mechanisms of action 43 are oxidative reactions and protein hydrolysis. 44 Oxygen and chlorine ions react with organic 45 material of microorganisms, such as Gram-posi- 46 tive and Gram-negative bacteria, fungi, viruses, 47 mycobacteria and yeast in the vegetative m 48 (spores). The oxidative reactions and protein 49 hydrolysis kill these microorganisms in both aer- 50 obic and anaerobic environments quickly, even at low concentrations. Because of the irrevers- Publication ibility of these processes, it is difficult these microorganisms to develop resistance to this 53 active principle Substantivity: Chloramine-T is strongly adsorbed 56 on hydroxyapatite (HA) due to the electrostatic 57 interaction between the positive ions of the cal- 58 cium in HA and the negative ions from chlorine. 59 This strong interaction provides time the anti- 60 microbial agent to act on the existing microor- 61 ganisms, releasing a proportional quantity of 62 active groups. 3. Stability at room temperature: The antiseptic agent 64 used in this system shows a remarkable stability 65 in aqueous solution even at high temperatures 66 (60 C). When exposed to environments with 67 organic material and microorganisms, the com- 68 pound remains 50% active after approximately h. Theree, the ions of Chloramine-T are 70 highly stable and remain active long periods Safety exposure in human beings: Chloramine-T 72 is used in several industries and other sectors: 73 personal hygiene, hospitals, water disinfection, 74 wastewater treatment, the food industry and oth- 75 ers. Within the specific dosage levels, it is neither 76 toxic nor irritating to the skin, eyes, mucosa, or, 77 particularly, the mouth. Technical inmation on 78 Chloramine-T indicates acute intoxication at 79 doses in the range of 50 to 1000 mg/kg and clas- 80 sifies the antimicrobial agent as not genotoxic or 81 mutagenic in the standard tests (Akzo Nobel, ) Based on the described characteristics of Chlora- 85 mine-t, it has been proposed to be added to denti- 86 frices (Pereira and Landi, 2007). Panzeri et al 87 (2008) evaluated an experimental dentifrice contain- 88 ing Chloramine-T total denture cleaning. They 89 showed that the experimental dentifrice was more 90 efficient in decreasing total denture biofilm than 91 water (control). Motivated these results, the 92 authors developed a dentifrice mulation contain- 93 ing Chloramine-T dental patients and preliminarily 94 tested it, with the null hypothesis stating that no dif- 95 ference in oral health indexes would be noticed when 96 comparing the experimental product containing 1% 97 Chloramine-T with a control. To evaluate this hypoth- 98 esis, the authors aim was to clinically compare the 99 experimental dentifrice with commercial-brand denti- 100 frice using dental plaque and sulcus bleeding 101 indexes (PI and SBI, respectively) Oral Health & Preventive Dentistry

3 1 MATERIALS AND METHODS 2 The research protocol was submitted to and 3 approved the Ethics Committee of the Ribeirão 4 Preto School of Dentistry, University of São Paulo, 5 Brazil, following the Helsinki Declaration. Table 1 Evaluated dentifrices Dentifrice Colgate Triple Action Colgate-Palmolive â Experimental dentifrice Publication Active agent Tirapelli et al 1500 ppm sodium monofluorophosphate and triclosan 1% Chloramine-T 6 Pre-study period: volunteer selection, 7 preparation, experimental design 8 Volunteer selection 9 All of the recruited subjects read and signed the 10 Terms of Inmation and Acceptance, which 11 explained in detail and in non-technical language 12 how the investigation would be permed to evalu- 13 ate two unnamed dentifrices 7 days and the free- 14 dom of acceptance or withdrawal at any time given to 15 the patients during the investigation. 16 Thirty patients from the School of Dentistry of 17 Ribeirão Preto were recruited according to the follow- 18 ing inclusion criteria: orally healthy individuals with a 19 non-zero PI and SBI; no periodontal disease (no prob- 20 ing depth exceeding 3.0 mm); age between 15 and years; no prosthetic or orthodontic appliances; 22 no systemic drugs or medications; normal appear- 23 ance of soft and hard oral tissues; and the presence 24 of at least 20 natural teeth. 25 Preparation 26 The single examiner (one of the clinician authors) of 27 the present study prepared the intra-oral exami- 28 nation, PI and SBI measurements a week bee 29 the beginning of the volunteer selection. The exam- 30 iner became familiar with the files the clinical 31 examinations and PI and SBI assessments during 32 consultations general patients from the dental 33 school where the present study was permed. It 34 was pre-determined that the same examiner would 35 select the patients and assess the PI, SBI baseline 36 and follow-up scores. 37 Experimental design 38 This was a clinical, fully randomised, double-blind 39 comparative study to evaluate the clinical efficacy 40 of an experimental dentifrice. A sample of 30 volun- 41 teers and a period of 7-day use of the experimental 42 product were decided, as the study was a preliminary 43 investigation of the experimental dentifrice. The iden- 44 tity of the dentifrices used (Table 1) was not dis- 45 closed to the patients or to the examiner clinician 46 assessing PI and SBI. Simple randomisation Fig 1 Illustration of the appearance of the dentifrices and tubes the double-blind experiment. (a) Similar consistencies of the dentifrices. (b) Identical white opaque numbered tubes in which the different (experimental and commercial-brand) dentifrices were inserted. allocated the 30 selected volunteers into two groups of 15 patients each. Each group was provided with one of the evaluated dentifrices. The experimental dentifrice was mulated and inserted into an opaque, numbered dentifrice tube. The commercial-brand dentifrice was removed from its original tube and inserted into an identical opaque, numbered dentifrice tube (Fig 1). The numbers of the tubes were randomly assigned. Only one member of the study staff had access to the inmation pertaining to which tubes contained which dentifrices. Clinical examination, PI and SBI assessments In the initial consultation, all selected volunteers underwent baseline clinical examinations in accordance with American Dental Association (ADA) guidelines to observe the eventual side effects of the dentifrices. The mucous membranes of the tongue, the hard and the soft palate, the gingiva, the Q Vol 8, No 4,

4 Tirapelli et al 1 muco-buccal folds, the inner surfaces of the cheeks 2 and sublingual space were examined. Normal or 3 abnormal appearance was recorded; as stated 4 above, only patients presenting normal appearances 5 these areas were included in the present study. 6 Supragingival plaque was assessed based on the 7 Turesky et al (1970) modification of the Quigley 8 and Hine (1962) procedure on buccal and lingual 9 surface of teeth 16, 11, 26, 36, 41 and 46. The 10 SBI, as proposed Muhlemann and Son (1971), 11 was scored at four sites: mesiobuccal, midbuccal, 12 distobuccal and midlingual. 13 Each patient received a white opaque dentifrice 14 tube that was identified the number labelled on 15 it. No new toothbrushes or oral hygiene guidance 16 was given to the patients. Written inmation about 17 the period of the present study was provided to the 18 volunteers to carry out the following instructions: 19 (1) no other dentifrice could be used; (2) oral mouth- 20 washes could not be used; (3) toothbrushes could 21 not be changed; and (4) the use of any other medi- 22 cine during the study period must be disclosed. 23 After 7-day use of the dentifrices, the same exam- 24 iner assessed the PI and SBI again. Clinical examina- 25 tion according to ADA guidelines was repeated, and 26 the normal or abnormal appearance of each oral tis- 27 sue was recorded. Publication 28 Statistical analyses 29 The scores obtained in the present study were 30 analysed using the Kruskal Wallis test, which is a 31 non-parametric test evaluating equality among 32 multiple groups considering ordinal scales and 33 pairwise comparisons. Dunn s post hoc test was 34 used to compare the difference between the sum 35 of the ranks of the two groups with the expected 36 average difference. The data were analysed using 37 Prism version 5.0 (GraphPad) Windows. 38 RESULTS 39 Twenty-nine of the selected volunteers returned 1 40 week after baseline examinations. The median age 41 of the patients was approximately 35 years; 67% 42 were females and 33% were males. The sample 43 included volunteers from the School of Dentistry of 44 Ribeirão Preto, University of São Paulo, Brazil, cho- 45 sen from among the patients attending the School s 46 dental clinic, students and office workers. It was 47 expected that the connection of all volunteers to 48 the University campus favoured their return after 1 49 week, as access to the study site was easy. No Fig 2 Graphic illustration of the SBI (a), the PI at buccal surfaces (b) and the PI at the lingual surfaces (c). Over each column are the median values obtained from the PI and SBI assessments. On the x-axis, 0 = baseline and 1 = 7-day use of the tested dentifrices. The level of statistical significance the comparisons between bee and after the use of the products is shown below each graph. abnormal appearance was observed in mucous membranes of the tongue, hard or soft palate, gingiva, muco-buccal folds, inner surfaces of the cheeks 50 4 Oral Health & Preventive Dentistry

5 1 or sublingual space after 1 week of using the denti- 2 frices. Figure 2 shows a graphical illustration of the 3 SBI and PI scores measured bee and after the 4 use of the dentifrices. 5 Baseline values PI and SBI were not different 6 between the two groups, showing that the random 7 distribution was effective. The PI scores diminished 8 after 1 week of use of both dentifrices, with statistical 9 significance the experimental dentifrice at buccal 10 (P < 0.01 versus baseline) and lingual sites 11 (P < 0.05) and the triclosan dentifrice at buccal 12 and lingual sites (P < 0.01). SBI score values 13 decreased significantly both experimental 14 and commercial-brand dentifrices compared to 15 baseline. 16 DISCUSSION 17 Given that the participation of the patients is impor- 18 tant improving oral health status (Ost et al, ), the search suitable instruments and prod- 20 ucts to make home oral hygiene more effective has 21 become an even more important challenge. The jus- 22 tification the use of chemical antimicrobials in the 23 mulation of dentifrices and other oral hygiene 24 products is twofold. First, the two most prevalent 25 oral diseases are caused bacteria. Second, it is 26 difficult to maintain people motivated to properly 27 clean their teeth consistently along their lives. In this 28 context, the chemical control must be complemen- 29 tary to the mechanical control of dental plaque. 30 Based on these facts, new proposals of dentifrice 31 mulations to control and prevent caries, gingivitis 32 and periodontal disease are often published. Some 33 of the well-known chemical agents have been pro- 34 posed in a few studies. For instance, Olympio et al 35 (2006) compared dentifrices with 1100 ppm NaF; 36 experimental 1100 ppm NaF plus 0.95% chlorhexi- 37 dine and experimental 0.95% chlorhexidine denti- 38 frices in terms of dental plaque, gingivitis, 39 bleeding, calculus and extrinsic enamel staining in 40 volunteers on fixed orthodontic therapy. Chlorhexi- 41 dine dentifrices significantly increased the mean 42 stain index, and dental calculus was not affected. 43 The products containing chlorhexidine were statisti- 44 cally better dental plaque, gingivitis and bleeding 45 scores. Slot et al (2007) observed the efficacy of a 46 chlorhexidine dentifrice gel, a regular fluoride- 47 containing dentifrice and mouthwash dentifrice in a 48 3-day non-brushing study. They concluded that the 49 application of 0.12% chlorhexidine dentifrice gel 50 was not different from the application of regular dentifrice regarding PI, and the most efficient in this Publication Tirapelli et al case was the mouthwash. Innovative proposals, such as the use of natural products in the dentifrice mulation the prevention and treatment of oral conditions, have been put th (Singh et al, 2007) or evaluated. Dentifrices containing Aloe vera extract, example, exhibited lower efficacy on the control of dental plaque and gingivitis compared with regular fluoride-containing dentifrices (Oliveira et al, 2008). In the present study, 1% Chloramine-T was evaluated in an experimental dentifrice the clinical indexes of plaque and sulcus gingival bleeding. To test the experimental dentifrice, it was compared with a gold-standard dentifrice containing triclosan. Triclosan (2,4,4 0 -trichloro-2 0 -hydroxydiphenyl ether) is a non-cationic antimicrobial agent that has been extensively used in consumer products, such as deodorants, soaps and dermatological preparations, and has recently been included in the list of oral health care products, such as dentifrices and mouthrinses. Clinical studies on human beings have shown it to be safe to use, causing no systemic or local side effects, no disturbance of the oral microbial ecology (Gaffar et al, 1994; Palomo et al, 1994) and reductions in dental plaque, gingivitis and calculus (DeSalva et al, 1989). Dentifrices containing triclosan show efficacy in clinical indexes of oral health (Lindhe et al, 1993; Triratana et al, 2002), and in the present study the volunteers who used the dentifrice containing triclosan experienced decreases in dental plaque and sulcus gingival bleeding. Chloramine-T was introduced as a disinfectant Dakin et al (1916), and since then, the product has come to be considered safe human use and is widely employed in various industries. Pitten and Kramer (1999) used Chloramine-T in mouthwash mulation, and Panzeri et al (2008) evaluated an experimental dentifrice containing Chloramine-T total denture cleaning on 60 subjects and showed a significant decrease in the amount of total denture biofilm when compared with a control. It has been suggested that Chloramine-T could be efficiently and safely used in dentifrice mulations. This possibility motivated us to mulate and study the efficacy of an experimental dentifrice containing Chloramine-T in dental patients. An experimental dentifrice containing Chloramine-T has produced decreased levels of Streptococcus mutans in total denture biofilms (Panzeri et al, 2008), but there are no previous reports on Chloramine-T among dental populations. As a preliminary investigation, the present study allocated 15 subjects who used the experimental product Chloramine-T 7 days. The 7-day use of the experimental product showed a significant reduction in dental plaque and Vol 8, No 4,

6 Tirapelli et al 1 sulcus bleeding scores. Furthermore, the new denti- 2 frice was shown to be safe, as no alterations in the 3 structures examined were observed. By comparing 4 the experimental and gold-standard dentifrice, it 5 was found that both reduced the PI significantly. 6 The significance level at which the triclosan denti- 7 frice decreased PI on lingual surfaces compared to 8 baseline (P < 0.01) was greater than that the 9 Chloramine-T dentifrice (P < 0.05). 10 With regard to the changes in the clinical indexes 11 in the present study, it is necessary to discuss 12 patient behaviour as a limitation of this investigation. 13 The study protocol deliberately excluded oral health 14 education the volunteers; nonetheless, the entire 15 study was carried out in a dental school, where the 16 volunteers were naturally exposed to an environment 17 with oral health motivation and inmation. Further- 18 more, taking into consideration the Hawthorne effect, 19 the improvements in oral health status are still likely 20 to have been caused the change in the dentifrices. 21 With regard to the methodology of the present 22 study, it may be concluded that no difference in PI 23 (at buccal sites) or SBI was observed when the 24 experimental dentifrice containing Chloramine-T and 25 the commercial-brand dentifrice containing triclosan 26 were used 7 days. Theree, the null hypothesis 27 of the present study was not rejected. Further, com- 28 parative studies involving other regular dentifrices, 29 control groups with no changes in oral hygiene hab- 30 its, long-term evaluations and induced gingivitis sam- 31 ples are required. 32 CONCLUSIONS 33 The experimental dentifrice containing 1% Chlora- 34 mine-t and one commercial-brand dentifrice contain- 35 ing triclosan had the same effect on clinical indexes 36 of oral health. 37 REFERENCES Akzo Nobel. 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